Regulation of embryonic patterning and adult stem cells of oral appendages

描述(申请人提供)：了解调控口腔外胚层附属物，如牙齿、味乳头和丝状乳头发育、构型和出生后更新的分子和细胞机制，并确定这些器官中的干细胞和祖细胞群，对于制定再生策略以修复先天性缺失或因疾病而缺失的牙齿至关重要；对于了解和治疗味觉障碍，包括放射治疗和小分子抗癌药物所导致的味觉障碍，以及描述口腔癌中可能受到失调的增殖控制至关重要。Wnt/-catenin信号通路在许多发育过程中都是必需的，在成体干细胞群体的增殖和自我更新中起着关键作用。小鼠的遗传学研究揭示了Wnt/-catenin信号在牙齿和味觉乳头形态发生的早期阶段的关键功能。信号在牙齿和味觉乳头发育开始之前被广泛激活，并逐渐被限制在牙齿和味觉前体发育的位置，确保牙齿和味觉器官的正确定位。根据我们的初步数据，我们假设Wnt信号的正确定位需要Wnt配体和分泌的Wnt抑制剂的竞争活性，这些竞争活性通过在空间上控制口腔上皮细胞的命运、运动和增殖来直接器官形成。 我们将使用小鼠胚胎口腔外植体的实时成像，这些外植体在Wnt激活的细胞或所有基底上皮细胞中特异地表达染色质定位的GFP，以及无处不在的膜番茄表达，以询问Wnt抑制剂的基因缺失或对牙齿发育至关重要的Wnt配体的丢失是否会导致细胞运动和/或增殖模式的改变。在成人生活中，舌丝状和味觉乳头不断更新，WNT10A突变的人类患者中的一部分表现为青春期口腔外胚层缺陷的发作，包括“光滑的舌头”。我们推测，WNT10A/-catenin信号控制着成体干细胞和前体细胞的增殖和/或存活，这些细胞是更新味觉和丝状乳头所必需的。为了测试这一点，我们将询问小鼠Wnt10a的缺失是否会影响连环蛋白信号转导和舌乳头前体细胞的增殖、存活或分化，并将命运映射到成年舌头中的Wnt反应细胞，以测试它们是否包括自我更新的前体细胞。为了确定Wnt信号是否对功能前体细胞的生存是必要的，我们将测试在去掉诱导剂后，丝状和味觉乳头对Wnt抑制因子Dkk1可诱导转基因表达的抑制是否可逆。这些实验将描述口腔中控制增殖和器官更新的机制，并将为设计再生策略提供重要信息。

DESCRIPTION (provided by applicant): Understanding the molecular and cellular mechanisms regulating the development, patterning and postnatal renewal of oral ectodermal appendages such as teeth, taste papillae and filiform papillae, and identifying stem and progenitor cell populations in these organs, is critical for developing regenerative strategies to replace missing teeth in cases of congenital absence or loss through disease; for understanding and treating disorders of taste, including those resulting from radiation therapy and small molecule anti-cancer drugs; and for delineating proliferation controls that may be dysregulated in oral cancers. The Wnt/ -catenin signaling pathway is necessary for many developmental processes and plays critical roles in the proliferation and self- renewal of adult stem cell populations. Genetic studies in mice reveal key functions for Wnt/ -catenin signaling at early stages of tooth and taste papilla morphogenesis. Signaling is activated broadly prior to the initiation of tooth and taste papilla development and gradually becomes restricted to sites of tooth and taste precursor development, ensuring correct positioning of tooth and taste organs. Based on our preliminary data, we hypothesize that proper localization of Wnt signaling requires competing activities of Wnt ligands and secreted Wnt inhibitors, and that these direct organ formation by spatially controlling the fates, movements and proliferation of oral epithelial cells. We will use live imaging of embryonic oral explants from mice that express chromatin-localized GFP specifically in Wnt-activated cells or in all basal epithelial cells, together with ubiquitous expression of membrane Tomato, to ask whether genetic deletion of Wnt inhibitors, or loss of a Wnt ligand important for tooth development, cause altered cell movements and/or patterns of proliferation. Filiform and taste papillae of the tongue are continuously renewed in adult life, an a subset of human patients with mutations in WNT10A presents with adolescent onset of oral ectodermal defects including "smooth tongues". We hypothesize that WNT10A/ -catenin signaling controls proliferation and/or survival of adult stem and progenitor cells required for renewal of taste and filiform papillae. To test this we will ask whether loss of Wnt10a in mice affects -catenin signaling and the proliferation, survival, or differentiation of tongue papilla progenitor cells and will fate map Wnt responsive cells in the adult tongue to test whether they include self- renewing progenitors. To determine whether Wnt signaling is necessary for survival of functional progenitors, we will test whether inhibition of filiform and taste papilla proliferaton upon inducible transgenic expression of the Wnt inhibitor DKK1 is reversible after removal of the inducing agent. These experiments will delineate mechanisms controlling proliferation and organ renewal in the oral cavity and will provide important information for designing regenerative strategies.